This course familiarizes students with the novel concepts being used to revamp regulatory toxicology in response to a breakthrough National Research Council Report “Toxicity Testing in the 21st Century: A Vision and a Strategy.” We present the latest developments in the field of toxicology—the shift from animal testing toward human relevant, high content, high-throughput integrative testing strategies. Active programs from EPA, NIH, and the global scientific community illustrate the dynamics of safety sciences.

Enseigné par

Lena Smirnova

Research Associate

Thomas Hartung

Professor

Transcription

In this section, we will talk about another epigenetic mechanism which called microRNA. And we will focus in this section on Biogenesis and functions of microRNAs. I will start from the very beginning. Back in 1958, when Francis Crick postulated his central dogma for molecular biology; DNA, one gene, one transcript, mRNA encode one protein. But a human genome project, and I quote, revealed that 90% of human genome is transcribed, 80% is biochemically functional, but only 1.5% of transcribed RNAs encode proteins. So, we have a lot of space in our genome for regulatory non-coding elements. This means that this scheme on the central molecular dogma may look a little bit different. A part of messenger RNA, we have tRNA and ribosomal RNA that are involved in translation. But recently, huge class of regulatory non-coding RNA molecules was discovered. The regulatory non-coding RNA which are longer than 200 nucleotides, called long non-coding RNAs, and which are smaller than 200 nucleotides, called small non-coding RNAs. This class of non-coding RNAs consist of several different RNAs. For example, small nuclear rRNA, small nuclear RNA, pvRNA or piRNA which expressed only in germ cells, snRNA which is responsible for RNA interference and microRNAs. MicroRNAs are most advanced studied class of small non-coding RNAs and I will focus today on these microRNAs. So, what are microRNAs? This is a class of small non-coding RNA molecules which are phylogenetically conserved. There are 22 nucleotide long, so they're really, really tiny, and they're produced from 70 nucleotide hairpin precursor. MicroRNAs binds to the site of imperfect complementarity in three prime untranslated region of messenger RNA target. And through this binding to the binding sites, they regulate mRNA translation. As long as microRNAs bind, messenger RNA cannot be translated to the protein. The first microRNA which was discovered was lin-4 link in C. elegans 1993. And the second microRNA followed only eight years later in 2001 and is called let-7. microRNAs were discovered in C. elegans by Victor Ambros Lab, who was started in heterochronic genes during the development of C. Elegans. On this slide, you can see different larva stages of development, L1, L2, L3, L4 and adult stage, and some heterochronic genes which are responsible for these development. In the larva stage 1, we have high expression of lin-14 and lin-28 genes. However, to be able to Transit's from L1 to L2 stage, those genes should be repressed. And Ambros group identified lin-4 as a repressor of those genes. However, Their mutant of lin-4 gene was not able to transmit from larva stage 1 to 2. And this was the first microRNA discovered, lin--4. Several years later, Ruvkun and his group identified another non-coding RNA which they called let-7. And this microRNA was regulated into transition from larva stage 4 to adult stage. In larva stage 4, we have high expressed lin-41 protein. And in order to block this gene, let-7 should be induced. And this lead to the transition to adult stage. However, let-7 mutant animals were not able to become adult animals. The microRNA field exploded after several laboratories revealed using cloning in bioinformatics techniques. There were hundreds of such microRNAs present in both plant and animal genomes. And as you can see on these histogram how the publications cited in PubMed and then miRBase entries increased over 10 years. In 2013, more than 20,000 entries were found in PubMed. And this number is still increasing. This slide shows you screenshot of microRNA Database called miRBase, which collect all microRNAs which were found. And now we are already by miRBase version 21. So, basically here you can perform the search for microRNA of your interest in different organism and see all information about these microRNA which is available. This cartoon will show you and guide you to the microRNA biogenesis which is a big complex. MicroRNAs are transcribed by a polymerase II into a long primary transcript called primary microRNA. This primary transcript is cleaved in nucleus by RNAs lead Drosha to 17 nucleotides stem loop hairpin pre-cursor or pre-microRNA. Next step, this pre-microRNA is exported from the nucleus to cytoplasm by Exportin5, where it's cleaved again by the RNase called Dicer. The mature microRNA, which is 22 nucleotides long, is then associated with their protein complex called microRNA induced silencing complex or miRISK. And together is this complex find its target to bind to the binding site and repress the translation. There are two different mechanisms how translation can be repressed. If microRNA bind with imperfect complementarity to Messenger RNA, it's just repressor translation. However, if microRNA binds to messenger RNA with perfect complementarity, it leads to a messenger RNA cleavage. So, how microRNA recognize their targets? Here's again two examples from C. elegans of lin-14 and lin-41 genes. This way you can see the 3-prime UTR of those genes. Blue lines show the binding sites of lin-4 microRNA and green lines show binding sites of let-7 microRNA. And the most important region in microRNA responsible for this binding is called seed region, which is nucleotide 2 to 8 in microRNA. In this seed region, which is highlighted in orange, the complementarity should be a 100% perfect and 3-prime area of microRNA can have some mismatches. This table shows some bioinformatical tools which were developed to predict microRNA targets. They have different algorithms which are based mainly on the conservation of microRNAs filler, complementarity especially as I already said in seed region and calculation of binding energy. This bioinformatic work revealed that more than 60% of all mammalian messenger RNA may be regulated by microRNAs. One microRNA may regulate more than a hundred targets. One target messenger RNA may have several binding sites for one or more microRNAs. microRNA regulate almost every cellular process in our body. Of course all this predicted microRNA-messenger RNA pairs should be experimentally validated because the bioinformatics prediction doesn't necessarily means that this connection is real. And this is an example of such validation based on our own research. The mlin gene lin-41 has predicted binding sites full at 7, shown here in red. And to validate that, we cloned 3-prime untranslated regions 3-prime UTR from these genes into the GFP vector, which means this is a plasmid which have fluorescent stock. And then we designed so-called mlin-41 sensor. And as a control, we imitated several places in the binding sites for let-7 and called this mutant control. Then we translated these sensor vectors in the cells which naturally express let-7. And as you can see here to the right, the upper corner cells which were translated to GFB Sensor are not green anymore because let-7 binds to their binding sites and pre-press the translation of GHB protein. However, the control cells with mutant binding sites where let-7 cannot any longer bind to the GHP messenger RNA remain green. So, this is the example of the validation of microRNA and messenger RNA targets and every single pair which is identified and predicted should be validated in a similar way. Taken in account that microRNAs regulates so many genes, it is not a surprise that they are involved in crucial processes in our body. MicroRNA function as a timer or switch regulating developmental timing. They decide when and where, during development, certain processes should be activated like cell proliferation or apoptosis, cell differentiation and specification. These fundamental functions suppose that microRNA could be involved in development of diseases. The best studied so far is the role of microRNA in cancer, where some microRNA, some act as a tumor suppressors and others have oncogenic activity. But microRNAs are of course involved in many other diseases such as neurodegenerative diseases; Parkinson's, Alzheimer, autoimmune diseases, inflammation, organ injury, metabolic diseases such as diabetes and several others. This slide show the example how microRNAs involved in cancer can be studied, what does it mean and how the therapeuticals can be developed based on this knowledge. There are several types of microRNAs in terms of cancer. Some of them are tumor suppressor, means that they regulate oncogens and some microRNA can be oncogens, means that they repress tumor suppressor genes. In case if tumor suppressor microRNA is downregulated, it leads to overexpression of oncogenic targets. And if oncogenic microRNA is overexpressed it leads to down regulation of tumor suppressor targets. In both cases, it will leads to cancer. And we can interfere in this stage by overexpression of tumor suppressor microRNAs or knockdown of the oncogenic microRNAs, and these can be used in cancer therapy. And several microRNAs are already in clinical trials. In this session, we talked about microRNA, its discovery, biogenesis and the function. In the next session I will focus on the role of microRNA in development.